FUNCTIONAL-PROPERTIES OF THE UPTAKE OF AMINES IN IMMORTALIZED PEPTIDERGIC NEURONS (TRANSPORT-P)

1 Most neurotransmitters are inactivated by uptake into presynaptic ne rve terminals and into glial cells. We recently provided evidence for uptake of amines in postsynaptic neurones. Uptake was evident at nanom olar concentrations of prazosin, but at concentrations of unlabelled p razosin greater than 10(-7) M, there was a further activation of uptak e, manifested by a paradoxical increase in accumulation of the radioli gand. We have now studied further characteristics of amine uptake in i mmortalised gonadotrophin-releasing hormone (GnRH) neurones. Control c ells included SK-N-SH neuroblastoma cells (which possess presynaptic t ype amine transporters) and non-neuronal (COS-7) cells. 2 [H-3]-prazos in bound to intact GnRH cells and was displaced by unlabelled prazosin in concentrations of 10(-9) to 10(-7) M. However, at higher concentra tions of unlabelled prazosin, there was an increase in apparent [H-3]- prazosin binding, as we had previously described. This paradoxical inc rease in accumulation of the radioligand was abolished by desipramine. 3 Desipramine had no effect on the association of prazosin with COS-7 cells. There was no paradoxical increase in accumulation of [H-3]-pra zosin in COS-7 cells, indicating that this effect requires the presenc e of a desipramine-blockable uptake process. 4 The increase in binding of the radioligand that was observed in the GnRH cells is not a gener al property of neuronal transporters; in SK-N-SH cells, there was no i ncrease in accumulation of (-)-[H-3]-noradrenaline in the presence of concentrations of unlabelled (-)-noradrenaline greater than 10(-7) M. 5 The uptake of prazosin and the increase in accumulation of [H-3]-pra zosin were abolished in the cold, indicating that this is an active, e nergy-requiring process. 6 Desipramine-sensitive uptake of prazosin wa s demonstrable in the GnRH cells in the absence of sodium. Further, th e Na+/K+-ATPase inhibitor, vanadate, abolished noradrenaline uptake in SK-N-SH cells but had no effect on prazosin uptake in GnRH cells. Thu s, the uptake of prazosin does not derive its energy from the sodium p ump. 7 Prazosin uptake was inhibited by the V-ATPase inhibitor bafilom ycin A(1), the H+/Na+ ionophore, monensin and the organic base, chloro quine, indicating that uptake derives its energy from a proton pump. I n contrast to other proton-dependent amine transporters, the uptake of prazosin was unaffected by reserpine. 8 Increasing extracellular pH d id not increase the uptake of prazosin into GnRH cells, indicating tha t it is unlikely to be due to non-specific diffusion and concentration of a lysosomotropic drug into intracellular acidic particles. 9 The u ptake of prazosin was unaffected by steroid hormones. 10 In COS-7 cell s transfected with alpha(1)-adrenoceptor cDNA, [H-3]-prazosin was disp laced by unlabelled prazosin without causing an increase in binding of the radioligand. This indicated that the increase in accumulation of the radioligand is unlikely to be due simply to some function of alpha (1)-adrenoceptors. 11 Thus, peptidergic neurones possess an uptake pro cess with properties that are distinguishable from known amine transpo rters.